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LB Media

LB is the most common media used to grow recombinant Escherichia coli (E. coli). LB media was named by Luria Bertani as "lysogeny broth" in deference to the research he was conducting on lysogeny in Escherichia coli. LB is commonly incorrectly referred to LB as Luria-Bertani media, Luria Broth or Lennox Broth. LB media is also used to culture a variety of facultative organisms.

An antibiotic is often added to the sterilized LB medium to select for cells that contain a specific genetic element such as a plasmid, a transposon or a gene disruption via an antibiotic resistance cassette. X-Gal (5-Bromo-4-Chloro-3-Indolyl-β-D-Galactoside) may be added to sterile LB medium when using the blue-white screen for plasmids containing the α-fragment of the β-galactosidase gene. IPTG, a lactose analog (isopropyl-β-D-thiogalactopyranoside) is added to induce expression of genes controlled by the lac promoter (the α-fragment is utilized in many plasmids in molecular cloning and the ω-fragment on the F' episome is in some strains of E. coli).

Components of LB Media:
One reason LB has been so popular is because it is simple to make, with only a few ingredients in the media: Tryptone, Yeast Extract and NaCl. Tryptone, a mixture of peptides formed by the digest of casein with the pancreatic enzyme trypsin, provides the source of nitrogen and carbon. Strains of E. coli that are derived from the K-12 strain, one of the most commonly used parental strains of E. coli used in molecular biology are deficient in B vitamin production. Yeast extract provides vitamins (including B vitamins) and certain trace elements. Sodium chloride provides sodium ions for transport and for osmotic balance.

Historical Background of LB:
LB is a nutritionally rich medium that has been used since the 1950's to culture Enterobacteriaceae and also for bacteriophage plaque assays. LB permits fast growth and good growth yields for many species. In 1951, Giuseppe Bertani developed LB to optimize plaque formation in a Shigella indicator strain of Enterobacteriaceae. Today, LB media is the most common media for growth of recombinant strains of Escherichia coli.

There are three common formulations of LB, which can be confusing as all three formulations are sometimes incorrectly referred to as LB Media. Bertani named LB for Lysogeny Broth.
It is common to (incorrectly) refer to LB as Luria-Bertani media, Luria Broth or Lennox Broth. This confusion is understandable given the history of Bertani, Luria and Lennox. Giuseppe Bertani was a member of the Salvador Luria lab at Indiana University when he formulated the original composition of LB media. Ed Lennox was a member of the Luria lab and worked with Bertani on some of the early experiments in lysogeny utilizing Shigella. Luria published a paper in 1955 in which he copied the original formulation of Bertani and LB is sometimes incorrectly attributed to Luria due to his scientific stature. That is, LB is often incorrectly referred to as Luria Broth.

The common alternate formulations of LB, known as LB-Miller, LB- Lennox and LB-Luria vary in the amount of sodium chloride (NaCl) in the media. The original formulation by Bertani contained: Bacto Tryptone 10g/L (1.0%), Yeast Extract 5g/L (0.5%), Sodium Chloride 10.0g/L (1.0%), pH adjusted to 7.0 with 1N NaOH and Glucose 1.0g/L (0.1%) to be added after autoclaving. Over time, the addition of Glucose has dropped out of all commonly used formulations of LB.

The original formulation of LB containing 1.0% NaCl (10.0g/L) is referred to as LB-Miller and it is the most common LB media used today. In 1955, Ed Lennox was studying mechanisms of DNA synthesis utilizing E. coli strains sensitive to osmotic stress so he altered the original formulation to contain half the salt concentration of the original formula to contain NaCl 5.0g/L (0.5%). This variation of LB media is properly referred to as LB-Lennox. LB-Lennox, with half the salt in LB-Miller (5.0g/L NaCl), is used for cultivation of E. coli used in salt-sensitive antibiotic selections, such as Zeiocin. A third formulation of LB with a final salt concentration of 0.5g/L NaCl (0.05%) has been used to isolate marine organisms such as Vibrio and this media is referred to as LB-Luria, in deference to the stature of Salvador Luria in microbiology.

Mechanisms of Growth in LB:
LB media was originally designed for growth of bacteria at low densities. It is known that growth of
E. coli in LB usually stops when the OD600 (optical density at 600nm) reaches approximately 2.0 under normal growth conditions (37oC, shaking at 250rpm), corresponding to approximately 0.6mg of E. coli (dry weight) per ml.
Exponential growth, a period of steady-state growth is estimated to end when OD600 is between 0.6 and 1.0. In 2007, D'Ari and colleagues undertook a comprehensive study of the growth characteristics of LB, looking specifically at the physiology of E. coli K-12, one of the most common strains utilized in molecular biology today.

D'Ari and colleagues noticed that K-12 cells grown in the LB-Miller version of LB with a final OD600 of 0.6 -1.0 are not always in the same physiological state. D'Ari confirmed earlier observations of a diauxic growth mode in LB and noted an end to exponential growth at an OD600 of 0.3, much earlier than commonly expected. It is known that E.coli is known to be unable to grow when the external pH exceeds 9.0 and it is not uncommon for LB media to have a pH close to 9.0, so D'Ari tried adjusting the pH of LB to no affect. D'ari found when glucose was added to the media, he was able to grow cultures to a final OD600 of 6.49, suggesting that the growth of E. coli in LB is carbon limited. As noted, the original formulation of LB by Bertani, contained 0.1% Glucose (1.0g/L) to be added after autoclaving. The work by D'Ari underscores that while LB is a good media for routine growth, it is not to be used for physiological studies where reproducibility is required.

Our experience in-house has found that for most strains of E. coli commonly utilized today in molecular cloning, there is no difference in growth characteristics, whether using LB-Miller (10.0g/L NaCl) or LB-Lennox (5.0g/L NaCl) formulations. However, it is important to use the appropriate media for selection utilizing salt-sensitive antibiotics such as Zeocin. LB-Lennox is most commonly used in this case.